Two Type III or cGMP-inhibited cyclic nucleotide phosphodiesterases (cGI PDE) isoenzyme subfamilies, cGIP1 and cGIP2, have been cloned from human (H) and rat (R) sources. They exhibit the domain organization common to all PDEs, with N-terminal regulatory regions and the domain (approximately 270 amino acids) conserved among all mammalian PDEs in the C-terminal portion. Deduced sequences of the conserved domains of cGIP1 and 2 are very similar to each other except within a sequence of 44 amino acids. This so-called cGI PDE "insertion" not only distinguishes cGI PDEs from other PDE families, but may identify catalytic domains of cGI PDE subfamilies. N- and C-terminal deletions were constructed by PCR, and recombinant cGI PDEs were characterized by Western blotting, kinetics, IC50 values of inhibitors, and photoaffinity labeling, to identify better the catalytic core of recombinant RcGIP1 and HcGIP2 isoforms, to define the relationship between structure and function of cGI PDEs, to compare their catalytic and pharmacological properties, and to demonstrate the cGMP-binding site in cGI PDEs. The results indicate that the cGI PDE catalytic core includes the approx. 270 amino acid domain conserved among all PDEs plus some additional upstream and downstream sequences. The isolated catalytic core exhibits properties similar to authentic cGI PDEs, i.e., high affinity for cAMP and cGMP, sensitivity of cGMP hydrolysis to inhibition by cAMP and cilostamide, sensitivity of cAMP hydrolysis to inhibition by cGMP, and insensitivity to inhibition by rolipram. RcGIP1 recombinants are less sensitive to inhibition by cGMP than are HcGIP2 recombinants. HcGIP2 recombinants exhibit a lower Km for cGMP than do RcGIP1 recombinants. Photolabeling with 32P cGMP was inhibited by cGMP, cAMP, and cilostamide.